Various calcium flux agonists are known in the art and have vastly different origins. For example, certain compounds act as ionophores and typically raise intracellular calcium levels by importing calcium ions into the cell, while other compounds raise intracellular calcium levels by increasing calcium secretion from intracellular stores like the endoplasmic reticulum and mitochondria.
Among various other known ionophores, calcimycin (A23187) and ionomycin are natural products (from the Gram+ bacteria Streptomyces chartreusensis and Streptomyces conglobatus, respectively) which were initially described as antibiotics decades ago. More specifically, both A23187 and ionomycin demonstrate direct antibiotic activity against a variety of potential microbial pathogens as was reported in U.S. Pat. No. 3,960,667 and U.S. Pat. No. 3,873,693. Unlike classical antibiotics (e.g., penicillin or tetracycline) that inhibit biochemical pathways specific to microbial proliferation such as bacterial cell wall synthesis or bacterial ribosome function, A23187 and ionomycin belong to a separate class of antibiotic compounds that bind divalent cations as substrates with relatively high specificity. For example, A23187 binding affinity is characterized by Mn2+>Ca2+=Mg2+>Sr2+>Ba2+ while ionomycin is characterized by Ca2+>Mg2+>Ba2+>Sr2+.
On the other hand, thapsigargin is a typical ER secretagogue and can be characterized as a sesquiterpene lactone. Thapsigargin is isolated from a plant (Thapsia garganica) and acts as a non-competitive inhibitor of various sarco/endoplasmic reticulum Ca2+ ATPases (SERCA). Thapsigargin:SERCA binding demonstrates an affinity constant in the low picomolar range and is toxic to both dividing and non-dividing cells. In animals, limited skin contact with thapsigargin can result in inflammation and chronic repetitive topical exposure can result in non-malignant papilloma formation when used in conjunction with a strong DNA damaging agent. In addition to thapsigargin (and structural analogs like thapsigargicin, etc.), other SERCA inhibitors include cyclopiazonic acid (CPA) and 2,5-di-tert-butylhydroquinone (DBHQ).
Difficult-to-treat skin infections represent an emerging public health concern for several reasons including the ever-increasing number of diabetic patients suffering from chronic skin ulcers, the presence of antibiotic resistant microbial flora (e.g., methicillin-resistant Staphylococcus aureus or MRSA), and the increasing frequency of outbreaks of necrotizing fasciitis (or “flesh-eating bacteria disease”). In addition to bacteria, many fungi and viruses can also cause significant infections of the skin. Although antibiotics remain the best treatment option for many of these disorders, it would be desirable to stimulate a patient's own cells and their associated functions to further improve patient recovery from ongoing infections and possibly even to stimulate long-term immunity against the offending organism to limit pathogenesis upon subsequent encounter.
In higher organisms, epithelial tissues including the skin serve as a critical barrier against pathogen-based, chemical, and physical insults. The epidermal layer of skin is comprised of keratinocytes, immune cells such as Langerhans cells and CD8+ T cells, Merkel cells and melanocytes. In addition to Langerhans cells, which are a subtype of dendritic cells (DC) responsible for disease surveillance, keratinocytes (which account for ˜95% of the total epidermal population) also serve as immune sentinels through their expression of various pattern recognition receptors such as members of the toll-like receptor (TLR) proteins, C-type lectin receptors (CLR), inflammasomes, etc.
Activation of these receptors in keratinocytes by their cognate ligands results in the release of both chemokines such as interleukin-8 (IL-8), CCL2, and CCL20 to recruit other immune cells as well as immune-regulating cytokines such as TGF-β and IL-10. Below the epidermis, the dermis is comprised of a larger variety of cell types including various subsets of CD4+ (Th1, Th2, Th17, etc) and non-classical (e.g. γδ, NK-, etc.) T lymphocytes, various antigen presenting cells (such as macrophages and dermal and plasmacytoid DC), mast cells and fibroblasts many of which express the various pattern recognition proteins described above. As such, both epidermal and dermal cells cooperate to prevent microbial invasion or other physical insults that could lead to significant disease.
The TLR proteins (TLR 1-10 in man) are type I membrane proteins which serve as pattern recognition receptors (PRR) for specific classes of ligands associated with disease and tissue homeostasis and as such are expressed on a variety of immune and non-immune cell types alike. As with all receptors, TLR signal transduction is triggered by ligand binding, and ligands may be grouped into pathogen-associated molecular patterns (or PAMP) and disease-associated molecular patterns (DAMP).
TLR-recognized PAMP include bacterial lipoproteins/lipopeptides, liposaccharides, flagellin, and unmethylated CpG DNA, fungal cell wall components (e.g. zymosan), and viral nucleic acids (dsRNA, ssRNA, and CpG DNA), while DAMPs are derived directly from the host, can occur in the absence of infection, and are recognized predominantly by only two members of the TLR family of proteins (either TLR2 and/or TLR4).
Once activated by their appropriate ligands, TLR initiate signaling cascades which serve both to limit the extent of infection/disease and to trigger tissue repair. With regards to the latter, TLR ligand recognition results in the up-regulation of antimicrobial activity and causes the activation/maturation of various immunological players to complete the destruction of the invading pathogen. For example, TLR activation can result in the release of reactive oxygen species (ROS), antimicrobial peptides, and upregulation of phagocytic function in innate cells such as macrophages, neutrophils, keratinocytes, etc. In addition to their role in combating disease, TLRs are also involved in tissue repair and regeneration as demonstrated in various models of tissue damage (including those induced by chemical, radiation, surgical, and infectious injury).
A further class of pattern recognition receptors is formed by the NOD-like receptor protein family, and includes NOD1 and NOD2 as the most prominent members. NOD1 and NOD2 are intracellular pattern recognition receptors, which are similar in structure to resistance proteins of plants, and mediate innate and acquired immunity by recognizing bacterial molecules containing D-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively. Following stimulation by their respective ligands, both NOD proteins interact with RIPK2 through respective recognition domains, which ultimately results in activation of the transcription factor NF-κB.
Previous efforts to characterize the response of intact skin to topical application of small molecule agonists of signal transduction (such as the protein kinase C agonist TPA/PMA or sustained calcium flux agonists like A23187, ionomycin, or thapsigargin) demonstrated a spectrum of downstream results. For example, topical application of the phorbol ester TPA caused vasodilation, microvascular permeability alterations, inflammatory cell recruitment, and the release of pro-inflammatory factors from various cell types. In contrast to PKC agonists, topical treatment with sustained calcium flux agonists (SCFA) resulted in skin inflammation and hyper-proliferation. On the other hand, exposure of cells to the TLR ligand (LPS) in the presence of relatively high quantities of a calcium ionophore (ionomycin) did not lead to any measurable immunostimulatory effect (Proc Natl Acad Sci USA. 2012 Jul. 10; 109(28): 11282-7). When applied individually at relatively high dosages, calcium ionophores and TLR ligands are known to stimulate differentiation or to activate dendritic cells as discussed in US 2012/0272700A1 and US 2013/0183343A1.
Therefore, while numerous compositions and uses for calcium flux agonists and/or ligands for TLR/NOD are known in the art, there is still a need to provide compositions and methods that provide improved immunomodulatory activity.